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Sunday, 29 June 2014

Cell's unique mutations used to trace history back to its origins in the embryo

Sunday, 29 June 2014

Researchers have developed new methods to trace the life history of individual cells back to their origins in the fertilised egg. By looking at the copy of the human genome present in healthy cells, they were able to build a picture of each cell's development from the early embryo on its journey to become part of an adult organ.

Reconstruction of early cell divisions of two

mouse embryos: Each white-filled large circle

represents an individual cell from a mouse

embryo and the unique combination of

mutations in its DNA. Each mutation is

represented by a number and those highlighted
in

yellow were acquired in the most recent cell

division. The smaller colour-filled circles are
the

final sets of cells taken from different parts
of

the mouse and there are an unknown number of

cell generations between each set of cells and
its

closest identifiable precursor cell. [DOI:

10.1038/nature13448]

During the life of an individual, all cells in the body develop mutations, known as somatic mutations, which are not inherited from parents or passed on to offspring. These somatic mutations carry a coded record of the lifetime experiences of each cell.

By looking at the numbers and types of mutations in a cell's DNA, researchers were able to assess whether the cell had divided a few times or many times and detect the imprints, known as signatures, of the processes of DNA damage and repair that the cells had been exposed to during the life of the individual. Furthermore, comparing each cell's mutations with those of other cells in the body enabled scientists to map out a detailed tree of development from the fertilised egg.

"With this novel approach, we can peer back into an organism's development," says Dr Sam Behjati, first author from the Wellcome Trust Sanger Institute.

"If we can better understand how normal, healthy cells mutate as they divide over a person's lifetime, we will gain a fundamental insight into what can be considered normal and how this differs from what we see in cancer cells."

The team looked at mouse cells from the stomach, small bowel, large bowel and prostate. The single cells were grown to produce enough DNA to be sequenced accurately. Eventually, single-cell sequencing technology will develop so that this type of experiment can be conducted using just one cell. However, the tiny amounts of DNA in single cells mean that mutation data are not currently precise enough to reconstruct accurate lineages.

The researchers recorded differences in the numbers of mutations in cells from the different tissues studied, likely attributable to differences in rates of cell division. Moreover, different patterns of mutation were found in cells from different tissues, suggesting that they have been exposed to different processes of DNA damage and repair, reflecting different lifetime experiences.

This experiment used healthy mice. If mutation rates are similar in human cells, these techniques could be used to provide an insight into the life histories of normal human cells.

"The adult human body is composed of 100 million million cells, all of which have originated from a single fertilised egg," says Professor Mike Stratton, senior author and Director of the Sanger Institute.

"Much more extensive application of this approach will allow us to provide a clear picture of how adult cells have developed from the fertilised egg. Furthermore, by looking at the numbers and types of mutation in each cell we will be able to obtain a diary, writ in DNA, of what each healthy cell has experienced during its lifetime, and then explore how this changes in the range of human diseases."

Mitochondria are cell organelles located within animal and human cells. They produce energy for the organism, possess their own genetic material - mitochondrial DNA (mtDNA) - and are transmitted exclusively by the mother. Depending on their activity and tasks, different numbers of mitochondria are present in a cell - usually a few hundred to a thousand per body cell.

Inherited mitochondrial disorders or so-called mitochondropathies occur in about one of 10,000 humans throughout the world. Diseases such as diabetes, stroke, cardiac defects, epilepsy, or muscle weakness may originate from mitochondrial defects. Inherited mitochondrial disorders have been incurable so far. Therefore, efforts are now being made to enable women with this disease to bear healthy children by means of nuclear transfer.

Mitochondria multiply at different rates

Jörg Burgstaller, a scientist and member of Gottfried Brem's research group at the Vetmeduni Vienna, has been working for several years on the genetics of mitochondria. It was known before that different types of mitochondria within a cell can proliferate at different rates. However, it was not known whether this is a singular phenomenon or if these cases occur more frequently.

Burgstaller investigated this in four newly bred mouse models which carried different mixtures of mitochondria whose DNA were related to each other to a differing extent.

This meant no health problem for the mice since all mtDNAs are were fully functional.

The outcome was: the more distantly two types of mitochondria within an egg cell were related, the more frequently a growth advantage was noted in favour of one of the two types of mitochondria. When two different mtDNAs were equally common in cells of an organ at the time of birth, one type was completely lost after a while. One mitochondria variant had thus achieved a growth advantage compared to the other variant and superseded the latter. This effect was almost non-existent in genetically very similar mitochondria within the cells; the ratio between the two types of mitochondria was not altered in that case.

The effect is of significance in reproduction medicine

Burgstaller's results may have effects on the planned introduction of the so called "Three-Parent Baby" in Great Britain. Experts take the cell nucleus of one human egg cell whose mitochondria have a defect and place it in an egg cell with "healthy" mitochondria. The baby resulting from this procedure has three parents, namely the mother whose cell nucleus is used, the mother whose mitochondria are involved, and the father whose sperm inseminated the egg cell.

However, this method raises the following problem: in every nuclear transfer, a small number of defective mitochondria are transferred into the healthy egg cell.

"So far it was believed that this minimal 'contamination' is of no consequence for the baby. However, our data show that the effect may have dramatic consequences on the health of the offspring. If the mitochondria of both mothers are genetically very different, it may have the same effects seen in the mouse model," says Burgstaller who developed the theory together with co-author Joanna Poulton, Professor of Mitochondrial Genetics at the John Radcliffe Hospital in Oxford.

"One mitochondrial type may be able to assert itself against the other. If the assertive one happens to carry the defective mtDNA, the benefit of the therapy would be jeopardized."

The solution to the "Three-Parent Baby"-problem

Burgstaller and his colleagues suggest the following solution to the problem: the mtDNA of both mothers, i.e. the donor of the nucleus and the donor of the mitochondria, should be analysed in advance and aligned to each other. So called "matching haplotypes" could prevent the dangerous effect. In the future the effect may even be utilized in a targeted manner to suppress defective mtDNA.

Tuesday, 17 June 2014

Tel Aviv University research uses new technique to uncover the building blocks of kidney regeneration

Tuesday, 17 June 2014

Doctors and scientists have for years been astonished to observe patients with kidney disease experiencing renal regeneration. The kidney, unlike its neighbour the liver, was universally understood to be a static organ once it had fully developed.

Now a new study conducted by researchers at Sheba Medical Center, Tel Aviv University and Stanford University turns that theory on its head by pinpointing the precise cellular signalling responsible for renal regeneration and exposing the multi-layered nature of kidney growth. The research, in Cell Reports, was conducted by principal investigators Dr. Benjamin Dekel of TAU's Sackler School of Medicine and Sheba Medical Center and Dr. Irving L. Weissman of Stanford University's School of Medicine, working with teams of researchers from both universities.

"We wanted to change the way people thought about kidneys – about internal organs altogether," said Dr. Dekel, who specializes in stem-cell research, genetics, and nephrology.

"Very little is known even now about the way our internal organs function at the single cell level. This study flips the paradigm that kidney cells are static – in fact, kidney cells are continuously growing, all the time."

Dr. Dekel began researching the subject three years ago while on sabbatical at Stanford University. While the laboratory experiments and stem cell research were conducted at Stanford, the results were analysed by researchers at TAU and Stanford.

According to Dr. Dekel, scientists knew kidney cells could reproduce outside the body, but the physiological process taking place inside the body at the single cell level was never explored. Uncovering that process became the focus of his efforts.

Dr. Dekel and his research team conducted a study using a "rainbow mouse" model developed at Stanford's Weissman lab, a mouse genetically altered to express one of four alternative fluorescent markers called "reporters" in each cell. The markers allowed researchers to trace cell growth in vivo — growth, they were surprised to find, that was sectional and multi-directional.

"We were amazed to find that renal growth does not depend on a single stem cell, but is rather compartmentalized," said Dr. Dekel.

"Each part of the nephron is responsible for its own growth, each segment responsible for its own development, like a tree trunk and branches – each branch grows at a different pace and in a different direction."

Using the rainbow mouse, the researchers were able to pinpoint a specific molecule responsible for renal cellular growth called the "WNT signal". Once activated in specific precursor cells in each kidney segment, the WNT signal results in robust renal cellular growth and generation of long branches of cells.

"Our aim was to use a new technique to analyse an old problem," said Dr. Dekel.

"No one had ever used a rainbow mouse model to monitor development of kidney cells. It was exciting to use these genetic tricks to discover that cellular growth was occurring all the time in the kidney – that, in fact, the kidney was constantly remodelling itself in a very specific mode."

Dr. Dekel and the research team are paving the way for novel cellular and molecular therapeutics to achieve human kidney regeneration and alleviate shortage of kidney organs for transplantation.

"This study teaches us that in order to regenerate the entire kidney segments different precursor cells grown outside of our bodies will have to be employed," he said.

"In addition, if we were able to further activate the WNT pathway, then in cases of disease or trauma we could activate the phenomena for growth and really boost kidney regeneration to help patients. This is a platform for the development of new therapeutics, allowing us to follow the growth and expansion of cells following treatment."

Scientists in the University of Connecticut's Technology Incubation Program have identified a novel approach to treating multiple sclerosis (MS) using human embryonic stem cells, offering a promising new therapy for more than 2.3 million people suffering from the debilitating disease.

The study was led by ImStem Biotechnology Inc. of Farmington, Conn., in conjunction with UConn Health Professor Joel Pachter, Assistant Professor Stephen Crocker, and Advanced Cell Technology (ACT) Inc. of Massachusetts. ImStem was founded in 2012 by UConn doctors Xiaofang Wang and Ren-He Xu, along with Yale University doctor Xinghua Pan and investor Michael Men.

"The cutting-edge work by ImStem, our first spinoff company, demonstrates the success of Connecticut's Stem Cell and Regenerative Medicine funding program in moving stem cells from bench to bedside," says Professor Marc Lalande, director of the UConn's Stem Cell Institute.

The research was supported by a $1.13 million group grant from the state of Connecticut's Stem Cell Research Program that was awarded to ImStem and Professor Pachter's lab.

"This new study moves us one step closer to a stem cell-based clinical product that could improve people's lives."

The researchers compared eight lines of adult bone marrow stem cells to four lines of human embryonic stem cells. All of the bone marrow-related stem cells expressed high levels of a protein molecule called a cytokine that stimulates autoimmunity and can worsen the disease. All of the human embryonic stem cell-related lines expressed little of the inflammatory cytokine.

Another advantage of human embryonic stem cells is that they can be propagated indefinitely in lab cultures and provide an unlimited source of high quality mesenchymal stem cells – the kind of stem cell needed for treatment of MS, the researchers say. This ability to reliably grow high quality mesenchymal stem cells from embryonic stem cells represents an advantage over adult bone marrow stem cells, which must be obtained from a limited supply of healthy donors and are of more variable quality.

"Ground-breaking research like this furthering opportunities for technology ventures demonstrates how the University acts as an economic engine for the state and regional economy," says Jeff Seemann, UConn's vice president for research.

The findings also offer potential therapy for other autoimmune diseases such as inflammatory bowel disease, rheumatoid arthritis, and type-1 diabetes, according to Xu, a corresponding author on the study and one of the few scientists in the world to have generated new human embryonic stem cell lines.

There is no cure for MS, a chronic neuroinflammatory disease in which the body's immune system eats away at the protective sheath called myelin that covers the nerves. Damage to myelin interferes with communication between the brain, spinal cord, and other areas of the body. Current MS treatments only offer pain relief, and slow the progression of the disease by suppressing inflammation.

"The beauty of this new type of mesenchymal stem cells is their remarkable higher efficacy in the MS model," says Wang, chief technology officer of ImStem.

The group's findings appear in the current online edition of Stem Cell Reports, the official journal of the International Society for Stem Cell Research. ImStem is currently seeking FDA approval necessary to make this treatment available to patients.

Rice University scientists apply new theory to learn how and why cells differentiate

Monday, 16 June 2014

How does a stem cell decide what path to take? In a way, it's up to the wisdom of the crowd.

The DNA in a pluripotent stem cell is bombarded with waves of proteins whose ebb and flow nudge the cell toward becoming blood, bone, skin or organs. A new theory by scientists at Rice University shows the cell's journey is neither a simple step-by-step process nor all random.

Peter Wolynes, left, and Bin Zhang of Rice

University tested their new method to analyse

large gene networks to begin to understand how

stem cells differentiate. Credit: Jeff
Fitlow/Rice

University.

Theoretical biologist Peter Wolynes and postdoctoral fellow Bin Zhang set out to create a mathematical tool to analyse large, realistic gene networks. As a bonus, their open-access study to be published this week by the Proceedings of the National Academy of Sciences helped them understand that the process by which stem cells differentiate is a many-body problem.

"Many-body" refers to physical systems that involve interactions between large numbers of particles. Scientists assume these many bodies conspire to have a function in every system, but the "problem" is figuring out just what that function is. In the new work, these bodies consist not only of the thousands of proteins expressed by embryonic stem cells but also DNA binding sites that lead to feedback loops and other "attractors" that prompt the cell to move from one steady state to the next until it reaches a final configuration.

To test their tool, the researchers looked at the roles of eight key proteins and how they rise and fall in number, bind and unbind to DNA and degrade during stem cell differentiation. Though the interactions may not always follow a precise path, their general pattern inevitably leads to the desired result for the same reason a strand of amino acids will inevitably fold into the proper protein: because the landscape dictates that it be so.

Wolynes called the new work a "stylized," simplified model meant to give a general but accurate overview of how cell networks function. It's based on a theory he formed in 2003 with Masaki Sasai of Nagoya University but now takes into account the fact that not one but many genes can be responsible for even a single decision in a cellular process.

An overview of the stem cell gene network gives

a sense of the complex process involved in cell

differentiation, as transcription factors and

protein complexes influence and loop back upon

each other. Rice University researchers found

that stem cell differentiation can be defined
as a

many-body problem as they developed a

theoretical system to analyse large gene

networks. Credit: Bin
Zhang/Rice University.

"This is what Bin figured out, that one could generalize our 2003 model to be much more realistic about how several different proteins bind to DNA in order to turn it on or off," Wolynes said.

A rigorous theoretical approach to determine the transition pathways and rates between steady states was also important, Zhang said.

"This is crucial for understanding the mechanism of how stem cell differentiation occurs," he said.

Wolynes said that because the stem cell is stochastic – that is, its fate is not pre-determined – "we had to ask why a gene doesn't constantly flip randomly from one state to another state. This paper for the first time describes how we can, for a pretty complicated circuit, figure out there are only certain periods during which the flipping can occur, following a well-defined transition pathway."

In previous models of gene networks, "Instead of focusing on proteins actually binding to DNA, they just say, 'Well, there's a certain high level of this protein or low level of that protein,'" Wolynes said.

"At first, that sounds easier to study because you can measure how much protein you've got. But you don't always know if it is bound. It has become increasingly clear that the rate of protein binding to DNA plays an important role in gene expression, particularly in eukaryotic systems."

The notion that many-body effects even existed in cells began in 1942 when British scientist C.H. Waddington established the idea of an epigenetic landscape for stem cells as a way to describe why pluripotent cells in embryos are destined to turn into bone, muscle and all the other parts of the body – but don't turn back. Waddington compared the cells' paths to marbles rolling to the bottom of a valley.

That concept rang true to Wolynes. His energy landscape theory has become key to understanding protein folding, although that theory sees the landscape as a funnel rather than a valley.

"Waddington said that as a cell develops into an embryo and beyond, it becomes many different kinds of cells," Wolynes said.

"Those cells might branch off and differentiate further, but they don't typically go back to the original state and start over.”

"His analogy – the idea of falling down through a valley – kicked around for a long time, but it was hard to make it mathematically precise. In his time, they didn't know about DNA," Wolynes said.

In both energy and epigenetic landscapes, Wolynes said, the steady state at the bottom is an attractor.

"It means wherever you start from, you end up attracted to that same place," he said.

"In genetic networks, things like steadily oscillating patterns can also be considered attractors."

Once biologists began to understand genetic switches in DNA, the whole picture became more complicated, he said.

"The landscape now has to incorporate the active parts of DNA that are trying to decide whether to turn this gene on or that gene off. In the '50s, we learned how genes made decisions on the basis of their production of proteins. These proteins then act back on the same genes in a kind of feedback loop."

The loops allow genes to remain active for far longer than it would take a protein simply to bind or unbind to a section of DNA. In the researchers' equations, the loops become attractors that help regulate transformation of the cell and can be mapped onto the many-body landscape.

Analysing the coupled dynamics of all these chemical reactions in a cell could be done by brute force, he said, but the computational cost would be enormous. So the Rice team decided to take a big-picture approach based on Wolynes' earlier work. It so happened that the resulting theoretical models of embryonic stem cells matched nicely with what experimentalists had seen in their studies.

For example, the models explained the fluctuations experimentalists had observed in the expression of a master regulator, a protein called Nanog, and its important role in maintaining a cell's pluripotency. Stem cells move from one steady state to the next on their journeys; in their calculations, they found a much higher level of Nanog gene expression in what they called SC1, the basic stem cell, than in SC2, a stem cell that had moved to the second steady state. This matched what experiments had measured, the researchers said.

"This is still just a beginning," Wolynes said.

"We're looking at embryonic stem cells now, but someday we want to treat the complete developmental program of organisms with hundreds of genes. We can see how these mathematics can scale up to that regime."

Friday, 13 June 2014

The offspring of chimpanzees inherit 90% of new mutations from their father, and just 10% from their mother, a finding which demonstrates how mutation differs between humans and our closest living relatives, and emphasises the importance of father's age on evolution.

In humans, each individual inherits, on average, about 70 new mutations from their parents. However, this number is influenced by paternal age such that older fathers tend to result in more mutations – in humans each extra year of age results in two extra mutations.

Mutation risk is linked to father's age because the sperm lineage in males keeps dividing, while females have all the eggs they are ever going to produce present at birth. Paternal age is an established risk factor in a number of disorders including schizophrenia and autism.

The study found that the number of new mutations inherited by chimpanzees from their parents is, on average, very similar to that in humans, but that the effect of the father's age is much stronger – each additional year of father's age results in three extra mutations.

The results suggest that sexual selection can influence the rate of evolution through its effect on the male mutation rate.

Professor Gil McVean, from the Wellcome Trust Centre for Human Genetics at the University of Oxford said:

"In humans, a father's age is known to affect how many new mutations he passes on to his children, and is also an established risk factor in a number of mental health disorders.”

"This study finds that in chimpanzees the father's age has a much stronger effect on mutation rate – about one and a half times that in humans. As a consequence, a greater fraction of new mutations enter the population through males, around 90 per cent, compared to humans, where fathers account for 75 per cent of new mutations."

In the study, Wellcome Trust-funded researchers sequenced the genomes of nine western chimpanzees from a three generation family living at the biomedical primate research centre in the Netherlands.

To establish the number of new mutations a child inherits researchers sequence children and their parents and compare the genetic sequence – any change in the sequence that doesn't exist in either parent genome is a new mutation. To find out which parent the mutation comes from you need to sequence members of the next generation of the family.

One explanation for this difference is that chimpanzees, as a result of their mating system, have evolved to produce many more sperm than humans – their testes are over three times the relative size of a human. This means there are likely to be more cycles of sperm production, increasing the opportunity for new mutations to emerge.

The authors suggest that more work needs to be done across other species to investigate the impact of mating behaviour on mutation rates and male mutation bias.

Tuesday, 10 June 2014

Researchers Use Human Stem Cells to Create Light-sensitive Retina in a Dish

Tuesday, 10 June 2014

These are rod photoreceptors (in green) within
a

"mini retina" derived from human iPS
cells in the

lab. Credit: Image courtesy of
Johns Hopkins

Medicine.

Using a type of human stem cell, Johns Hopkins researchers say they have created a three-dimensional complement of human retinal tissue in the laboratory, which notably includes functioning photoreceptor cells capable of responding to light, the first step in the process of converting it into visual images.

"We have basically created a miniature human retina in a dish that not only has the architectural organization of the retina but also has the ability to sense light," says study leader M. Valeria Canto-Soler, Ph.D., an assistant professor of ophthalmology at the Johns Hopkins University School of Medicine.

She says the work, reported online June 10 in the journal Nature Communications, "advances opportunities for vision-saving research and may ultimately lead to technologies that restore vision in people with retinal diseases."

Like many processes in the body, vision depends on many different types of cells working in concert, in this case to turn light into something that can be recognized by the brain as an image. Canto-Soler cautions that photoreceptors are only part of the story in the complex eye-brain process of vision, and her lab hasn't yet recreated all of the functions of the human eye and its links to the visual cortex of the brain.

"Is our lab retina capable of producing a visual signal that the brain can interpret into an image? Probably not, but this is a good start," she says.

The iPS cells are adult cells that have been genetically reprogrammed to their most primitive state. Under the right circumstances, they can develop into most or all of the 200 cell types in the human body. In this case, the Johns Hopkins team turned them into retinal progenitor cells destined to form light-sensitive retinal tissue that lines the back of the eye.

Using a simple, straightforward technique they developed to foster the growth of the retinal progenitors, Canto-Soler and her team saw retinal cells and then tissue grow in their petri dishes, says Xiufeng Zhong, Ph.D., a postdoctoral researcher in Canto-Soler's lab. The growth, she says, corresponded in timing and duration to retinal development in a human foetus in the womb. Moreover, the photoreceptors were mature enough to develop outer segments, a structure essential for photoreceptors to function.

Retinal tissue is complex, comprising seven major cell types, including six kinds of neurons, which are all organized into specific cell layers that absorb and process light, "see," and transmit those visual signals to the brain for interpretation. The lab-grown retinas recreate the three-dimensional architecture of the human retina.

"We knew that a 3-D cellular structure was necessary if we wanted to reproduce functional characteristics of the retina," says Canto-Soler.

"But when we began this work, we didn't think stem cells would be able to build up a retina almost on their own. In our system, somehow the cells knew what to do."

When the retinal tissue was at a stage equivalent to 28 weeks of development in the womb, with fairly mature photoreceptors, the researchers tested these mini-retinas to see if the photoreceptors could in fact sense and transform light into visual signals.

They did so by placing an electrode into a single photoreceptor cell and then giving a pulse of light to the cell, which reacted in a biochemical pattern similar to the behaviour of photoreceptors in people exposed to light.

Specifically, she says, the lab-grown photoreceptors responded to light the way retinal rods do. Human retinas contain two major photoreceptor cell types called rods and cones. The vast majority of photoreceptors in humans are rods, which enable vision in low light. The retinas grown by the Johns Hopkins team were also dominated by rods.

Canto-Soler says that the newly developed system gives them the ability to generate hundreds of mini-retinas at a time directly from a person affected by a particular retinal disease such as retinitis pigmentosa. This provides a unique biological system to study the cause of retinal diseases directly in human tissue, instead of relying on animal models.

The system, she says, also opens an array of possibilities for personalized medicine such as testing drugs to treat these diseases in a patient-specific way. In the long term, the potential is also there to replace diseased or dead retinal tissue with lab-grown material to restore vision.

Thursday, 5 June 2014

Case Western Reserve University research team discovers 'seeds' of stem cells' development

Thursday, 05 June 2014

Case Western Reserve researchers have discovered landmarks within pluripotent stem cells that guide how they develop to serve different purposes within the body. This breakthrough offers promise that scientists eventually will be able to direct stem cells in ways that prevent disease or repair damage from injury or illness. The study and its results appear in the June 5 edition of the journal Cell Stem Cell.

Pluripotent stem cells are so named because they can evolve into any of the cell types that exist within the body. Their immense potential captured the attention of two accomplished faculty with complementary areas of expertise.

“We had a unique opportunity to bring together two interdisciplinary groups,” said co-senior author Paul Tesar, PhD, Assistant Professor of Genetics and Genome Sciences at CWRU School of Medicine and the Dr. Donald and Ruth Weber Goodman Professor.

"We have exploited the Tesar lab’s expertise in stem cell biology and my lab’s expertise in genomics to uncover a new class of genetic switches, which we call seed enhancers,” said co-senior author Peter Scacheri, PhD, Associate Professor of Genetics and Genome Sciences at CWRU School of Medicine.

“Seed enhancers give us new clues to how cells morph from one cell type to another during development."

The breakthrough came from studying two closely related stem cell types that represent the earliest phases of development — embryonic stem cells and epiblast stem cells, first described in research by Tesar in 2007.

“These two stem cell types give us unprecedented access to the earliest stages of mammalian development,” said Daniel Factor, graduate student in the Tesar lab and co-first author of the study.

“Stem cells are touted for their promise to make replacement tissues for regenerative medicine,” she said.

“But first, we have to understand precisely how these cells function to create diverse tissues.”

Enhancers are sections of DNA that control the expression of nearby genes. By comparing these two closely related types of pluripotent stem cells (embryonic and epiblast), Corradin and Factor identified a new class of enhancers, which they refer to as seed enhancers. Unlike most enhancers, which are only active in specific times or places in the body, seed enhancers play roles from before birth to adulthood.

They are present, but dormant, in the early mouse embryonic stem cell population. In the more developed mouse epiblast stem cell population, they become the primary enhancers of their associated genes. As the cells mature into functional adult tissues, the seed enhancers grow into super enhancers. Super enhancers are large regions that contain many enhancers and control the most important genes in each cell type.

“These seed enhancers have wide-ranging potential to impact the understanding of development and disease,” said Stanton Gerson, MD, Asa & Patricia Shiverick and Jane Shiverick (Tripp) Professor of Hematological Oncology and Director of the National Center for Regenerative Medicine at Case Western Reserve University.

Proteins are responsible for the vast majority of the cellular functions that shape life, but like guests at a crowded dinner party, they interact transiently and in complex networks, making it difficult to determine which specific interactions are most important.

Directed
Network Wiring, a new method to

simplify
the study of protein networks, is

illustrated.
Credit: Shohei Koide/University of

Chicago.

Now, researchers from the University of Chicago have pioneered a new technique to simplify the study of protein networks and identify the importance of individual protein interactions. By designing synthetic proteins that can only interact with a pre-determined partner, and introducing them into cells, the team revealed a key interaction that regulates the ability of embryonic stem cells to change into other cell types. They describe their findings June 5 in Molecular Cell.

"Our work suggests that the apparent complexity of protein networks is deceiving, and that a circuit involving a small number of proteins might control each cellular function," said senior author Shohei Koide, PhD, professor of biochemistry and molecular biophysics at the University of Chicago.

For a cell to perform biological functions and respond to the environment, proteins must interact with one another in immensely complex networks, which when diagrammed can resemble a subway map out of a nightmare. These networks have traditionally been studied by removing a protein of interest through genetic engineering and observing whether the removal destroys the function of interest or not. However, this does not provide information on the importance of specific protein-to-protein interactions.

To approach this challenge, Koide and his team pioneered a new technique that they dub "directed network wiring." Studying mouse embryonic stem cells, they removed Grb2, a protein essential to the ability of the stem cell to transform into other cell types, from the cells. The researchers then designed synthetic versions of Grb2 that could only interact with one protein from a pool of dozens that normal Grb2 is known to network with. The team then introduced these synthetic proteins back into the cell to see which specific interactions would restore the stem cell's transformative abilities.

"The name, 'directed network wiring,' comes from the fact that we create minimalist networks," Koide said.

"We first remove all communication lines associated with a protein of interest and add back a single line. It is analysis by addition."

Despite the complexity of the protein network associated with stem cell development, the team discovered that restoring only one interaction — between Grb2 and a protein known as Ptpn11/Shp2 phosphatase — was enough to allow stem cells to again change into other cell types.

"We were really surprised to find that consolidating many interactions down to a single particular connection for the protein was sufficient to support development of the cells to the next stage, which involves many complicated processes," Koide said.

"Our results show that signals travel discrete and simple routes in the cell."

Koide and his team are now working on streamlining directed network wiring and applying it to other areas of study such as cancer. With the ability to dramatically simplify how scientists study protein interaction networks, they hope to open the door to new research areas and therapeutic approaches.

"We can now design synthetic proteins that are far more sophisticated than natural ones, and use such super-performance proteins toward advancing science and medicine," he said.